Paradigm shifting studies in the monarch butterfly (Danaus plexippus) have enhanced our view of the evolution and function of CRYPTOCHROME (CRY) proteins in animals. The butterfly's clock mechanism posits two CRY proteins as critical for circadian timing, giving rise to a NOVEL clockwork. Thus, in the same species, the two functionally distinct CRYs can be analyzed. In monarchs, a Drosophila-like CRY, designated CRY1, functions as a likely circadian photoreceptor, while a vertebrate-like CRY, designated CRY2, appears to function as the major transcriptional repressor of the clockwork transcriptional feedback loop. The analysis of distinct CRY function has been further aided by a monarch cell line (DpN1 cells), which expresses a light-driven clock, in which both monarch CRY1 and CRY2 function;no other light-sensitive insect cell line has been described. The Specific Aims of this proposal will expand our knowledge of the unique properties of the CRY proteins by defining distinct CRY mechanisms of action in lepidopterans (butterflies and moths) using molecular, biochemical, genetic, and behavioral approaches. Because of the parallel, complementary nature of circadian clock discoveries between flies and mammals, which can now be extended to lepidopterans, our overriding hypothesis is that further analysis of the two families of animal CRY proteins that are represented in insects will advance our fundamental understanding of animal clock mechanisms. The Specific Aims will 1) employ an RNAi screen in DpN1 cells to discover novel components of the insect CRY1 light-signaling pathway;2) use a proteomics approach in DpN1 cells to identify novel interactors within the insect CRY2/CLK:CYC transcriptional complex, utilizing tandem affinity purification and co-immunoprecipitation to identify additional CRY2-, CLOCK- and CYCLE-interacting proteins;and 3) define in vivo the critical clockwork function of insect CRY2 by targeted gene inactivation in the commercial silkworm Bombyx mori using a zinc finger nuclease strategy. Our major goal is to define the molecular mechanisms of CRY function in the animal clockwork. The proposed studies are innovative and provide an integrated approach at defining differential insect CRY mechanisms of action. The results will also increase our fundamental understanding of clockwork function from an evolutionary perspective and demonstrate lepidopterans to be a useful model in which to study animal clock mechanisms. Understanding the molecular clock should increase our knowledge of how clock gene mutations contribute to psychopathology. Likewise, such understanding should lead to new strategies for pharmacological manipulation of the human clock to improve the treatment of jet lag and shift-work ailments, and of clock-related sleep and psychiatric disorders. PUBLIC HEALTH RELEVANCE Understanding the molecular clock, the focus of this proposal, should increase our knowledge of how clock gene mutations contribute to psychopathology and some sleep disorders. Likewise, such understanding should lead to new strategies for pharmacological manipulation of the human clock to improve the treatment of jet lag and shift-work ailments, and of clock-related sleep and psychiatric disorders.